NBSGW Mice Research Articles

Developing a Humanized Murine Model for Co-Transplantation of Acute Myeloid Leukemia

Humanized mouse models and primary disease xenografts have produced significant translational insight into the biology of the bone marrow (BM) niche and aided drug discovery in acute myeloid leukemia (AML). To enable the study of cell-cell interactions between hematopoietic stem and progenitor cells (HSPCs) and AML as an emerging source of drug resistance, we describe two complementary approaches to humanized AML xenotransplantation models. One strategy utilizes the sequential transplantation of humanized hCD34+ cells followed by human AML cells. Recipient NBSGW mice (NOD.Cg-Kit W-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ), known for high rates of medullary engraftment, were conditioned with 15mg/kg busulfan followed by injection with either granulocyte colony-stimulating (G-CSF)-mobilized or BM-derived human CD34+ HSPCs. Flow cytometric analysis of the BM following injection of hCD34+ cells revealed efficient BM engraftment by both G-CSF- and BM- derived hCD34+ cells, demonstrated by BM human chimerism (hCD45%) of 63.7±10.0% by 8-weeks post-injection. Further BM hCD45 subtype analyses revealed B-cell (hCD45+ hCD19+) dominant repopulation (78.3±4.48%), followed by myeloid cell (hCD45+ hCD33+) repopulation (18.12±4.19%). T-cell repopulation was predictably minimal (0.008±0.01%). At 9- and 15- weeks post hCD34+ cells injection, GFP-expressing MOLM-14 AML cells (MOLM-14-GFP; 1E5, 1E6, or 2E6 cells) were injected. Flow cytometric analyses of MOLM-14-GFP BM chimerism up to 21 days post-injection showed complete absence of MOLM-14-GFP cells in the BM with no significant changes in overall hCD45 chimerism and hCD45 subtype composition compared to a PBS-injected control. These observations are consistent with a potential role for residual healthy hematopoietic cells in suppressing AML engraftment in the BM, previously shown in congenic studies. To test this hypothesis, we developed a second humanized AML model designed to investigate immunotherapeutic approaches for AML, where AML blasts are engrafted first, then followed by a human immune cell challenge. FA-AML1 or Kasumi-1 AML cells were injected into NSG mice (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) at 8 weeks of age. Injected mice were monitored for AML engraftment by HLA-DR expression in peripheral blood (PB). Engraftment occurred 6-12 weeks post-injection. After AML engraftment, we reconstituted a human immune system in the mice with a one-time injection of peripheral blood mononuclear cells (PBMCs), confirmed by flow cytometry for CD3+ T-cells (14.7±9.2%), CD33+ myeloid cells, and CD19+ B-cells. AML burden in the PB decreased 5-10% upon PBMC engraftment. In this model, a PB burden of <10% AML engraftment confers durable leukemic remission, whereas at >60% AML engraftment, leukemic cells persist in the periphery for over 100 days. Flow cytometric analysis demonstrated immune cell numbers peak in week 1 post-injection (30%), then decrease by week 4 to less than 1% in both the BM and PB, but persist at this low level for over 100 days. Altogether, these models confirm the ability of human hematopoietic cells to actively suppress leukemic engraftment in the BM microenvironment. More broadly, we demonstrate the development of two complementary humanized AML co-transplantation models that can attain dynamic ranges of human immune cell and AML cell engraftment to facilitate preclinical therapeutic development studies.

Therapeutic Gene Editing of HSCs Ex Vivo without in Vitro Culture Avoids Genotoxicity, Simplifies Procedures, and Preserves Efficiency and Stemness

Therapeutic gene editing of HSCs promises to cure inherited blood disorders. However, current ex vivo gene editing protocols may be associated with genotoxic potential and complex in vitro cell manufacturing procedures. Base and prime editing reduce double strand breaks (DSBs) but are generally less efficient and might have unique genotoxic potential (e.g. untargeted deaminase activity). Here we test the hypothesis that therapeutic gene editing with targeted endonucleases disrupting HbF-repressing elements for b-hemoglobinopathies may be made safer, simpler, and at least as effective by focusing the editing on nondividing cells. Genotoxic risks of DSBs appear to be concentrated in cycling cells. For example, Cas9-induced DSBs can result in long deletions at the on-target site that appear to be mainly the result of unidirectional extensive resection selectively active in S/G2 cell cycle phases1. In addition, DSBs must be resolved prior to mitosis to prevent chromosome mis-segregation leading to copy number variation and cytoplasmic micronuclei2,3. We measured the cell cycle state of mobilized PB CD34+ HSPCs immediately after collection, or after 24 or 48 hours of culture with cytokines (SCF, TPO, and FLT3L each at 100 ng/ml). 100% of cells were in G0/G1 at 0 hours, 94.6%±1.5% at 24 hours, and 76.7%±2.6% at 48 hours. We electroporated CD34+ HSPCs with 0, 24, or 48 hours of cytokine culture before performing 3xNLS-HiFi-SpCas9:sgRNA ribonucleoprotein (RNP) electroporation targeting the BCL11A erythroid enhancer. We employed a flow cytometry assay to quantify micronuclei in edited HSPCs. The fraction of micronuclei was increased in cells electroporated with Cas9:sgRNA RNP following 24 or 48 hours of cytokine stimulation but not in unstimulated cells. We performed ddPCR on sorted nuclei and micronuclei using assays specific to distal chromosome 2p (telomeric to the BCL11A cleavage site) and to chr1 as a reference autosome. We found ~9-fold enrichment of chr2p in micronuclei after 24 or 48 hours of pre-stimulation cytokine culture prior to gene editing, but strikingly did not observe any enrichment of chr2p in micronuclei in cells edited without pre-stimulation culture, suggesting that chromosome mis-segregation and unwanted downstream consequences could be avoided by editing quiescent HSCs (Fig. 1A). In addition, we developed ddPCR assays to measure genome editing outcomes after editing the BCL11A enhancer with paired cleavages (at the +58 and +55 enhancers) separated by 3-kb. These assays measure: 1) programmed 3-kb deletions, 2) programmed 3-kb inversions, or 3) short indels or unedited alleles. Any missing signals (genomic copies fewer than found on a reference nontargeted autosome) may represent long deletions or rearrangements. Editing cells without pre-stimulation culture was associated with undetectable missing signals (inferred long deletions and rearrangements) as compared to editing after 24 or 48 hours of pre-stimulation culture. We compared our standard protocol of 24 hours of pre-stimulation cytokine culture and 24 hours of post-electroporation cytokine culture to a protocol in which CD34+ HSPCs were electroporated immediately after thawing and then cryopreserved 15 minutes after electroporation, without any cytokine culture, before infusion to NBSGW mice. After 16 weeks, HbF levels were potently induced to similar levels after gene editing in recipients of HSPCs without and with cytokine culture (Fig. 1B). Overall human engraftment was similar for all groups, as was multilineage repopulation. Gene edit frequencies were similar for recipients of HSPCs without and with cytokine culture. We observed comparable editing and engraftment potency of HSPCs without as compared to with cytokine culture across mobilization type (G-CSF or plerixafor), donor disease status (healthy and SCD), and sgRNA target sequence. Together, these results suggest that editing HSPCs ex vivo without in vitro culture: avoids unintended on-target genotoxicity potential, including production of micronuclei, long deletions, and rearrangements; simplifies protocols, eliminating time, cost, and complexity; and preserves gene editing efficiency and HbF induction and HSC repopulation potency. Editing nondividing cells selectively could be a straightforward modification to improve ex vivo editing as well as to advance future efforts for safe and potent in vivo editing of HSCs. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Base Editing for Therapeutic Induction of Fetal Hemoglobin in β-Thalassemia

β-thalassemia, one of the most common inherited genetic disorders worldwide, is caused by more than 300 different mutations in the β-globin locus (HBB), all of which result in quantitative deficiency of β-globin protein with severe anemia and reduced or absent HbA (α2β2). Additionally, free α-globin protein forms toxic intracellular inclusions resulting in hemolysis of mature red blood cells, maturation arrest and apoptosis of erythroid precursors. The only approved cure for severe β-thalassemia is allogeneic hematopoietic stem cell transplantation. However, many patients lack ideal histocompatible donors and immune complications such as graft rejection and graft-versus-host disease can occur. These problems can be avoided by "gene therapies” in which the patient's own hematopoietic stem cells (HSCs) are isolated, genetically modified ex vivo and reinfused after administration of bone marrow conditioning to facilitate engraftment of the modified cells. Most β-thalassemia mutations can be treated by genetic manipulations that de-repress fetal-expressed γ-globin genes (HBG1 and HBG2) to induce fetal hemoglobin production (HbF, α2γ2) postnatally. Thus, Cas9 disruption of an erythroid enhancer in the γ-globin repressor gene BCL11A shows promising results in an ongoing clinical trial. However, multiple other strategies to induce HbF exist and the best one is not known. Compared to conventional Cas9 nuclease, base editors offer theoretical advantages, including the ability to create precise base pair substitutions without requiring genotoxic double strand DNA breaks. We used the adenine base editor ABE8e to generate the naturally occurring γ-globin -175 A>G variant, which causes hereditary persistence of fetal hemoglobin (HPFH), a benign genetic condition that alleviates co-inherited β-thalassemia. We edited human CD34+ hematopoietic stem and progenitor cells (HSPCs) from two individuals with severe transfusion dependent β-thalassemia by electroporating ribonucleoprotein (RNP) complexes of ABE8e-NG protein + single guide RNA (sgRNA) designed to install the γ-globin -175 A>G variant, followed by in vitro erythroid differentiation. On-target editing rates were 48-53% for donor 1 (n=3 replicates) and 22.9% for donor 2 (n=1). Analysis of burst forming unit erythroid (BFU-E) colonies revealed at least one on-target edit in 78% of donor 1 HSPCs and 63% of those from donor 2. Reverse phase HPLC of erythroid progeny showed increased β-like/α globin protein ratios: 0.92 ± 0.04 vs 0.53 ± 0.03 in unedited controls (p<0.0001) for donor 1, and 0.8 vs 0.5 in unedited controls for donor 2. Quantitative RT-PCR showed 6-8-fold increased γ-globin mRNA in the ABE-treated β-thalassemia cells. Ion exchange HPLC analysis of erythroid cell lysates showed reductions in free α-globin levels after ABE treatment: from 21.5 ± 2% of total globin protein in unedited cells to undetectable levels after editing in donor 1 and from 18% to 2.5% for donor 2. Maturation to the reticulocyte stage was also increased in base edited cells: 14.6 ± 2% vs 3.3 ± 1% in unedited controls for donor 1 (P<0.0001); and 26% vs 6.9% in unedited controls for donor 2. Reduction in apoptosis (37.4 ± 2.2% vs 23.6 ± 1.5% in unedited control cells, p=0.0003) and reactive oxygen species measured by DCFDA (MFI: 690 ± 97 vs 266 ± 9 in unedited control cells, p=0.009) was also seen in the ABE edited groups. To study editing outcomes in vivo, CD34+ HSPCs from donor 2 were treated with RNP and transplanted into immunodeficient NBSGW mice. After 16-18 weeks, human donor-derived cells were isolated from mouse recipient bone marrow and analyzed (n=4 mice per group). On-target editing frequencies in CD235a+ erythroblasts and CD34+ hematopoietic stem/progenitors were 17.5 ± 5% and 19.5 ±3%, respectively, compared to 22.9% in the input CD34+ cells. Similar to what we observed after in vitro differentiation, base edited erythroid cells generated in vivo exhibited improved globin chain balance with β-like/α globin protein ratios of 0.86 ± 0.12 vs 0.69 ± 0.04 in unedited controls, (p=0.04), reduced apoptosis (16.8 ± 4.2 % vs 26.6 ± 2.9 % in unedited control cells, p=0.004) and enhanced maturation into enucleated reticulocytes (14.4 ± 4.7 % vs 2.87 ± 1.4 % in unedited control cells, p=0.005). Our study provides proof of concept that ABE8e generation of the γ-globin -175 A>G HPFH variant in β-thalassemic HSCs represents a new therapeutic approach for β-thalassemia.

Intraosseous delivery of platelet-targeted factor VIII lentiviral vector in humanized NBSGW mice

AbstractWe previously showed that intraosseous (IO) delivery of factor VIII (FVIII, gene F8) lentiviral vector (LV) driven by the megakaryocyte-specific promoter Gp1bα (G-F8-LV) partially corrected the bleeding phenotype in hemophilia A (HemA) mice for up to 5 months. In this study, we further characterized and confirmed the successful transduction of self-regenerating hematopoietic stem and progenitor cells (HSPCs) in treated mice. In addition, secondary transplant of HSPCs isolated from G-F8-LV–treated mice corrected the bleeding phenotype of the recipient HemA mice, indicating the potential of long-term transgene expression following IO-LV therapy. To facilitate the translation of this technology to human applications, we evaluated the safety and efficacy of this gene transfer therapy into human HSPCs. In vitro transduction of human HSPCs by the platelet-targeted G-F8-LV confirmed megakaryocyte-specific gene expression after preferential differentiation of HSPCs to megakaryocyte lineages. Lentiviral integration analysis detected a polyclonal integration pattern in G-F8-LV–transduced human cells, profiling the clinical safety of hemophilia treatment. Most importantly, IO delivery of G-F8-LV to humanized NBSGW mice produced persistent FVIII expression in human platelets after gene therapy, and the megakaryocytes differentiated from human CD34+ HSPCs isolated from LV-treated humanized mice showed up to 10.2% FVIII expression, indicating efficient transduction of self-regenerating human HSPCs. Collectively, these results indicate the long-term safety and efficacy of the IO-LV gene therapy strategy for HemA in a humanized model, adding further evidence to the feasibility of translating this method for clinical applications.

The CCR5 Gene Edited CD34+CD90+ Hematopoietic Stem Cell Population Serves as an Optimal Graft Source for HIV Gene Therapy.

Transplantation of allogenic hematopoietic stem and progenitor cells (HSPCs) with C-C chemokine receptor type 5 (CCR5) Δ32 genotype generates HIV-1 resistant immune cells. CCR5 gene edited autologous HSPCs can be a potential alternative to hematopoietic stem cell transplantation (HSCT) from HLA-matched CCR5 null donor. However, the clinical application of gene edited autologous HSPCs is critically limited by the quality of the graft, as HIV also infects the HSPCs. In this study, by using mobilized HSPCs from healthy donors, we show that the CD34+CD90+ hematopoietic stem cells (HSCs) express 7-fold lower CD4/CCR5 HIV receptors, higher levels of SAMHD1 anti-viral restriction factor, and possess lower susceptibility to HIV infection than the CD34+CD90- hematopoietic progenitor cells. Further, the treatment with small molecule cocktail of Resveratrol, UM729 and SR1(RUS) improved the in vivo engraftment potential of CD34+CD90+ HSCs. To demonstrate that CD34+CD90+ HSC population as an ideal graft for HIV gene therapy, we sort purified CD34+CD90+ HSCs, treated with RUS and then gene edited the CCR5 with single sgRNA. On transplantation, 100,000 CD34+CD90+ HSCs were sufficient for long-term repopulation of the entire bone marrow of NBSGW mice. Importantly, the gene editing efficiency of ~90% in the infused product was maintained in vivo, facilitating the generation of CCR5 null immune cells, resistant to HIV infection. Altogether, CCR5 gene editing of CD34+CD90+ HSCs provide an ideal gene manipulation strategy for autologous HSCT based gene therapy for HIV infection.

Knock out of CD123 or CLL-1 By CRISPR-Cas9 Editing from Human Hematopoietic Stem Cell Transplants Provide New Possibilities for Increasing Therapeutic Index and Safety for AML Treatment

▪Acute myeloid leukemia (AML) is a clonal disorder of hematopoiesis and the most common form of acute leukemia in adults. Most patients with AML relapse despite intensive chemotherapy. Allogeneic hematopoietic cell transplant (HCT) has become the standard of care for patients with intermediate or adverse genetics, with >3,500 transplants performed annually in the US. However, leukemia relapse post-HCT occurs in ~40% of these patients with a 2-year survival rate at <20%, necessitating new approaches to reduce relapse and improve overall outcomes.Antigen-specific immunotherapies require cell surface antigens to be uniquely expressed on cancer cells to minimize “on-target, off-tumor” toxicity. CD123 and CLL-1 are highly expressed on normal myeloid cells, thus impeding the use of therapies targeting these antigens due to myelotoxicity. To circumvent such toxicity, we aim to create CD123 or CLL-1 negative human hematopoietic stem and progenitor cells (hHSPCs) for HCT such that this can be combined with subsequent use of targeted therapy against these antigens to prevent post-HCT relapse. Here, we present the pre-clinical evaluation of engineered hHSPCs, derived from mobilized peripheral blood of healthy donors, where CD123 or CLL-1 proteins were ablated by CRISPR/Cas9 gene editing. We have identified highly efficient guide RNAs that result in >80% on-target editing and can achieve greater than 90% biallelic gene knockout (KO). Deep sequencing followed by hybrid capture of up to 1000 potential off-target sites predicted to contain a maximum of 5 mismatches revealed minimal to no detectable off-target editing events. CD123 or CLL-1 KO hHSPCs showed >85% cell viability post-editing. Loss of CD123 or CLL-1 protein did not impact the differentiation of hHSPCs into granulocytic, monocytic, or erythroid lineages in vitro. Additionally, myeloid cells derived from CD123 or CLL-1 KO hHSPCs also retained their function, demonstrating similar phagocytotic capacity and the production of inflammatory cytokines in response to TLR agonists compared to unedited control cells. Importantly, CD123 or CLL-1 KO hHSPCs xenotransplanted into NSG or NBSGW mice showed no defect in long-term engraftment (human chimerism in NBSGW mice 16 weeks post transplant: 92.3±5.1% for CD123 KO, 91.2±4.1% for CLL-1 KO, and 93.4±3.1% unedited hHSPC engrafted mice, n=15 each). This in vivo model confirmed no observable impact on multilineage differentiation after CD123 or CLL-1 deletion either, supported by equal distribution of 10 hematopoietic lineages between groups. Bone marrow analyses revealed persistence of high gene editing frequency after 16 weeks (71±10% from CD123 KO and 86±6% from CLL-1 KO engrafted mice, n=15 each, compared to original 83% input CD123 KO and 86% input CLL-1 hHSPCs). Sorted subpopulations, including granulocytes, monocytes, dendritic cells, and mast cells, from the bone marrow similarly retained high levels of editing. Together these data suggest that there was no counter-selection against CD123 or CLL-1 KO cells, no outgrowth of any cells with particular edits or hematopoietic lineages to warrant tumorigenic concerns. Most notably, proof-of-concept experiments showed that cells depleted of CD123 or CLL-1 protein are indeed protected from the cytotoxicity of CD123 or CLL-1 targeted CAR-T cells.In conclusion, we demonstrate that CD123 or CLL-1 negative human HSPCs can successfully carry out functional hematopoiesis that is resistant to CD123 or CLL-1 targeted therapies. Our findings provide a next-generation HCT strategy that supports the safe and effective use of antigen-directed immunotherapy treatments for patients with AML. DisclosuresLuo: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Angelini: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Krishnamurthy: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Lisle: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Isik: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Ghdossi: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Cummins: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Pettiglio: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Hazelbaker: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Ge: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Tavares: Vor Biopharma: Ended employment in the past 24 months. Nikam: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Paik: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Lydeard: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Lin: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company. Chakraborty: Vor Biopharma: Current Employment, Current equity holder in publicly-traded company.

Preclinical Development of EDIT301, an Autologous Cell Therapy Comprising AsCas12a-RNP Modified Mobilized Peripheral Blood-CD34 + Cells for the Potential Treatment of Transfusion Dependent Beta Thalassemia

Beta thalassemia is one of the most common recessive hematological disorders in the world with more than 200 mutations identified to date. These mutations reduce or completely abrogate beta globin expression. As beta globin pairs with alpha globin to form adult hemoglobin (HbA, α2β2), reduced or absent beta globin results in excessive alpha globin chains, which form toxic aggregates. These aggregates cause maturation blockade and premature death of erythroid precursors, and hemolysis of red blood cells (RBC), leading to varying degrees of anemia. Patients with the most severe form of beta thalassemia, namely beta thalassemia major, are transfusion-dependent, i.e., requiring life-long RBC transfusions accompanied by the burden of iron chelation therapy.EDIT-301 is an experimental autologous cell therapy in which CD34 + cells are genetically modified to promote gamma globin expression. EDIT-301 is currently in clinical development for sickle cell disease, and IND enabling stage for transfusion-dependent beta thalassemia (TDT). Gamma globin decreases the alpha to beta globin chain imbalance in beta thalassemia by pairing with the over-abundant alpha globin chains to form fetal hemoglobin (HbF, α2γ2). Gamma globin induction, and consequently HbF induction, for EDIT-301 is achieved through AsCas12a ribonucleoprotein (RNP)-mediated editing of the distal CCAAT box region of the HBG1 and HBG2 promoters, where naturally occurring hereditary persistence of fetal hemoglobin (HFPH) mutations exist. We chose this target over BCL11A based on previous preclinical data demonstrating that BCL11A editing reduces erythroid output in NBSGW mice. An engineered AsCas12a RNP edits the HBG1 and HBG2 promoter distal CCAAT box with high efficiency and specificity. We have previously shown that on-target editing of >80% was achieved in mobilized peripheral blood (mPB) CD34 + cells from normal donors with no detectable off-target editing both at research scale and at clinical manufacturing scale. Edited normal donor CD34 + cells led to long-term, polyclonal, multilineage engraftment without lineage skewing in immunocompromised mice and sustained robust HbF production in their erythroid progeny.To test whether EDIT-301 may be an efficacious therapy for TDT, mPB CD34 + cells from individuals with TDT were electroporated with the engineered AsCas12a RNP targeting the HBG1 and HBG2 promoters. AsCas12a RNP edited mPB CD34 + cells from individuals with TDT as efficiently as CD34 + cells from normal donors. Importantly, EDIT-301 has the potential to address the underlying pathophysiology of TDT, i.e., the maturation blockade and premature death of erythroid precursors. Erythroid differentiation of edited beta thalassemia CD34 + cells showed significant improvement in erythroid maturation and health. Specifically, ~70% edited erythroblasts reached late erythroblast stage compared to ~53% unedited erythroblasts; ~56% edited erythroid cells underwent terminal maturation and enucleated compared to ~28% of unedited erythroid cells; and non-viable erythroblasts decreased from ~33% to ~22% after editing. The improved erythropoiesis was accompanied by significantly increased total hemoglobin content per cell. These data strongly support that editing of the HBG1 and HBG2 promoter CCAAT box using engineered AsCas12a RNP can reverse the dyserythropoiesis associated with beta thalassemia and increase the hemoglobin production.In summary, we have provided strong preclinical data supporting the development of EDIT-301 for the treatment of TDT. Edited mPB CD34 + cells retained their ability to engraft without lineage skewing, resulted in robust HbF induction long-term, improved erythropoiesis, and increased hemoglobin content in TDT erythroid cells. These data support that a single administration of EDIT-301 may have the potential to safely and effectively reverse dyserythropoiesis and ameliorate anemia in individuals with TDT long-term. Clinical studies to demonstrate the safety and efficacy of EDIT-301 in the treatment of TDT are currently being planned. DisclosuresSousa: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Janoudi: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. deDreuzy: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Shearman: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Zhang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company. Chang: Editas Medicine: Current Employment, Current equity holder in publicly-traded company.

Bone Marrow Stem Cells from Patients with Myelodysplastic Syndrome (MDS) Engraft and Migrate in-between the Humanized Niches in Immuno-Compromised Mouse Models

IntroductionMyelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized mainly by ineffective hematopoiesis. Xenograft models of MDS in immunodeficient mice have been instrumental in improving our understanding of clonal origin and evolution of this disease (Mian & Rouault-Pierre et al., Nat Commun. 2015; Rouault-Pierre & Mian et al Haematologica 2016; Leukemia, 2017); however, the low level of engraftment generally observed in these mouse models makes them impractical for testing therapeutic strategies. We used a 3D humanized scaffold in vivo system to test the MDS stem cell engraftment as well as study the cross talk between MDS-Initiating cells (MDS-ICs) and their niche.MethodologyIn this study bone marrow (BM) cells from 31 patients (27 MDS cases covering all WHO subtypes, 1 CMML, 1 MDS/MPD and 2 sAML) were studied. Initially, whole-exome sequencing was performed on all cases. Colony-forming assay was used to assess the colony forming ability of BM CD34+ cells from each patient. Mesenchymal stromal cells (MSCs) were isolated from the BM of the patient samples. We then used 3 immunodeficient mouse models (NSG, NSG-SGM3 and NBSGW) to study the engraftment kinetics and clonal evolution of the transplanted cells in gelatin-based 3D scaffolds coated with patient MSCs (RARS/RCMD, autologous; RAEB, autologous/allogenic).ResultsBM MSCs obtained from the MDS patients showed variable expansion levels, with the ability to differentiate into the adipogenic lineage whereas the osteogenic differentiation was highly aberrant.Next, 0.5x106 to 3x106 patient (n=31) mononuclear cells (MNCs T cell depleted) were seeded into 3D-scaffolds prior to implantation in NSG-SGM3 and/or NSG mice. We observed persistent long-term engraftment of hCD45+ cells ranging from 0.05% to 80% (up to 18 weeks). Importantly, 74% of the cases generated an engraftment level of >20% hCD45+ cells. Similar engraftment levels were observed in mice humanized scaffolds that were injected with BM cells from MDS/MPD, CMML and sAML (hCD45+ 10%, 22% and 86%, respectively). No difference in the hCD45+ cell engraftment between the patients with low-risk or high-risk MDS was observed.Remarkably, our 3D humanized scaffolds generated multi-lineage engraftment, with mice that received BM cells from RAEB cases yielding higher myeloid engraftment compared to RARS and RCMD (RARS hCD33+ 44%; RCMD hCD33+ 46.6%; RAEB hCD33+ 65%). Furthermore, transplantation of BM cells from 5 patients into NBSGW mice gave rise to engraftment levels of hCD45+ cells similar (hCD45+ cells, up to 40%) to the NSG and NSG-SGM3 mice. The MDS cells recovered from the mice maintained their genotypic characteristics, as demonstrated by the presence of the mutations that remained largely unchanged between the pre- and post-transplanted samples.Interestingly, Immuno-staining of the scaffolds revealed colonization by the host murine vasculature (mCD31+ endothelial cells). In line with this finding, mouse hematopoietic cells were also found to have migrated into the implanted humanized scaffolds. Surprisingly, we did not observe hCD45+ cell engraftment in the bone marrow of any of the xenotransplanted mouse strains (NSG or NSG-SGM3 mice with or without Irradiation and non-irradiated NBSGW mice). However, we demonstrated that MDS stem cells migrate by implanting a supplementary scaffold into NSG-SGM3 and NBSGW mice that was only coated with MDS-MSCs. Notably, these MDS-MSC seeded scaffolds were colonized by the circulating human MDS cells.Finally, we tested whether the cells recovered from the primary engrafted 3D humanized scaffolds would be able to generate secondary transplants. Our data shows that these cells were able to engraft in the secondary mice implanted with humanized 3D scaffolds.DiscussionOur model provides evidence that MDS BM cells are highly dependent on humanized MSC niche, confirming the importance of the cross-talk between MDS-ICs and MDS niche. Our data demonstrates that MDS stem cells do migrate out of the 3D Scaffold but require their “own niche” for colonization. Thus, our in vivo 3D model system, can be reliably used to better dissect the cross-talk between MDS stoma and MDS-ICs. This model requiring relatively low number of human MNCs, provides a robust preclinical assessment tool to study clonal evolution as well as to test therapeutic strategies on the MDS clonality prior to treatment of MDS patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Enhanced HbF reactivation by multiplex mutagenesis of thalassemic CD34+ cells in vitro and in vivo

AbstractThalassemia or sickle cell patients with hereditary persistence of fetal hemoglobin (HbF) have an ameliorated clinical phenotype and, in some cases, can achieve transfusion independence. Inactivation via genome editing of γ-globin developmental suppressors, such as BCL11A or LRF/ZBTB7A, or of their binding sites, have been shown to significantly increase expression of endogenous HbF. To broaden the therapeutic window beyond a single-editing approach, we have explored combinations of cis- and trans-editing targets to enhance HbF reactivation. Multiplex mutagenesis in adult CD34+ cells was well tolerated and did not lead to any detectable defect in the cells' proliferation and differentiation, either in vitro or in vivo. The combination of 1 trans and 1 cis mutation resulted in high editing retention in vivo, coupled with almost pancellular HbF expression in NBSGW mice. The greater in vivo performance of this combination was also recapitulated using a novel helper-dependent adenoviral-CRISPR vector (HD-Ad-dualCRISPR) in CD34+ cells from β-thalassemia patients transplanted to NBSGW mice. A pronounced increase in HbF expression was observed in human red blood cells in mice with established predominant β0/β0-thalassemic hemopoiesis after in vivo injection of the HD-Ad-dualCRISPR vector. Collectively, our data suggest that the combination of cis and trans fetal globin reactivation mutations has the potential to significantly increase HbF both totally and on a per cell basis over single editing and could thus provide significant clinical benefit to patients with severe β-globin phenotype.

Development of LT-HSC-Reconstituted Non-Irradiated NBSGW Mice for the Study of Human Hematopoiesis In Vivo.

Humanized immune system (HIS) mouse models are useful tools for the in vivo investigation of human hematopoiesis. However, the majority of HIS models currently in use are biased towards lymphocyte development and fail to support long-term multilineage leucocytes and erythrocytes. Those that achieve successful multilineage reconstitution often require preconditioning steps which are expensive, cause animal morbidity, are technically demanding, and poorly reproducible. In this study, we address this challenge by using HSPC-NBSGW mice, in which NOD,B6.SCID IL-2rγ -/-KitW41/W41 (NBSGW) mice are engrafted with human CD133+ hematopoietic stem and progenitor cells (HSPCs) without the need for preconditioning by sublethal irradiation. These HSPCs are enriched in long-term hematopoietic stem cells (LT-HSCs), while NBSGW mice are permissive to human hematopoietic stem cell (HSC) engraftment, thus reducing the cell number required for successful HIS development. B cells reconstitute with the greatest efficiency, including mature B cells capable of class-switching following allogeneic stimulation and, within lymphoid organs and peripheral blood, T cells at a spectrum of stages of maturation. In the thymus, human thymocytes are identified at all major stages of development. Phenotypically distinct subsets of myeloid cells, including dendritic cells and mature monocytes, engraft to a variable degree in the bone marrow and spleen, and circulate in peripheral blood. Finally, we observe human erythrocytes which persist in the periphery at high levels following macrophage clearance. The HSPC-NBSGW model therefore provides a useful platform for the study of human hematological and immunological processes and pathologies.

Aging of human hematopoietic stem cells is linked to changes in Cdc42 activity.

In this study, we characterize age-related phenotypes of human hematopoietic stem cells (HSC). We report increased frequencies of HSC, hematopoietic progenitor cells and lineage negative cells in the elderly but a decreased frequency of multi-lymphoid progenitors. Aged human HSC further exhibited a delay in initiating division ex vivo though without changes in their division kinetics. The activity of the small RhoGTPase Cdc42 was elevated in aged human hematopoietic cells and we identified a positive correlation between Cdc42 activity and the frequency of HSC upon aging. The frequency of human HSC polar for polarity proteins was, similar to the mouse, decreased upon aging, while inhibition of Cdc42 activity via the specific pharmacological inhibitor of Cdc42 activity, CASIN, resulted in re-polarization of aged human HSC with respect to Cdc42. Elevated activity of Cdc42 in aged HSC thus contributed to age-related changes in HSC. Xenotransplant, using NBSGW mice as recipients, showed elevated chimerism in recipients of aged compared to young HSC. Aged HSC treated with CASIN ex vivo displayed an engraftment profile similar to recipients of young HSC. Taken together, our work reveals strong evidence for a role of elevated Cdc42 activity in driving aging of human HSC, and similar to mice, this presents a likely possibility for attenuation of aging in human HSC.

Adenosine Base Editing of γ-Globin Promoters Induces Fetal Hemoglobin and Inhibit Erythroid Sickling

Rare variants in the γ-globin (HBG2 and HBG1) promoters cause sustained postnatal expression of fetal hemoglobin (HbF, α2γ2) in red blood cells (RBCs). This benign condition is termed hereditary persistence of fetal hemoglobin (HPFH). Individuals with HPFH variants are protected from β-hemoglobinopathies including sickle cell disease and β-thalassemia. Our group and others have used CRIPSR/Cas9-mediated non-homologous end joining to generate HPFH-like insertion-deletion (indel) mutations in the γ-globin promoter. However, simultaneous double-stranded breaks (DSBs) in the tandem duplicated γ-globin genes can result in loss or inversion of the intervening genetic material and/or chromosomal rearrangements. More generally, Cas9-associated DSBs can elicit a cytotoxic DNA repair response leading to cell death or evoke p53 loss with malignant transformation. Base editor (BE) proteins represent a promising approach to install precise nucleotide substitutions without DSBs. Adenosine base editors (ABEs), consisting of catalytically impaired Cas9 fused to a modified adenosine deaminase, create targeted A:T-to-G:C mutations. Here we describe the use of ABEs to recapitulate naturally occurring HPFH variants in hematopoietic stem cells (HSCs). We electroporated ABE7.10-single guide (sg) RNA ribonucleoprotein (RNP) complex into mobilized peripheral blood CD34+ hematopoietic stem and progenitor cells (HSPCs) to recreate 3 different HPFH variants in the HBG1/2 promoters (-198 T&amp;gt;C, -175 T&amp;gt;C and -113 A&amp;gt;G). Measured editing frequency was maximal on day 10 after electroporation and transferred to erythroid differentiation media. 20% editing efficiency was observed for the -198 site, 58% for -175 and 50% for -113. Indel frequencies were &amp;lt;2% at each of the three sites, reflecting a low rate of DSBs. Fetal hemoglobin levels in erythroid cells generated in vitro from A base-edited CD34+ HSPCs were 26±4% (-198 T&amp;gt;C), 60±10% (-175 T&amp;gt;C), and 42±7% (-113 A&amp;gt;G) versus14±2% in unedited control cells. Base editing at the -175 site in sickle cell disease (SCD) donor CD34+ HSPCs resulted in the induction of HbF to 55% in erythroid progeny compared to 6% in controls. After exposure to hypoxia (2% oxygen), reticulocytes generated from -175 T&amp;gt;C-edited CD34+ HSPCs exhibited sickling rates of 24%, compared to 52% in controls. Thus, creation of this variant, which generates a de novo binding site for the transcriptional activator TAL1, reactivates erythroid cell HbF to levels that inhibit sickle hemoglobin polymerization and cell sickling. To assess base editing in HSCs, we used ABE RNP to modify the -175 site in SCD donor CD34+ HSPCs, followed by transplantation into NBSGW mice. The editing frequency in CD34+ HSPCs before transplantation was ~30% and declined to approximately 20% in bone marrow-repopulating donor cells at 16 weeks post-transplantation. Editing frequencies were similar in CD34+ donor cell-derived myeloid, erythroid, and B cells, indicating that hematopoietic differentiation was not altered. Bone marrow erythroblasts derived from base-edited and control CD34+ HSPCs exhibited similar maturation profiles and enucleation. Erythroblasts generated in vivo from SCD patient HSPCs exhibited 32±2% HbF compared to unedited controls (4±1%) (n=4, P&amp;gt;0.0001). Our studies provide proof of concept that adenosine base editors can be used therapeutically for β-hemoglobinopathies. Specifically, generation of the -175 T&amp;gt;C HPFH mutation in patient HSCs followed by autologous transplantation represents a new therapeutic approach for SCD and β-thalassemia. Disclosures Yen: Beam Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Sharma:Spotlight Therapeutics: Consultancy; Magenta Therapeutics: Other: Research Collaboration; CRISPR Therapeutics, Vertex Pharmaceuticals, Novartis: Other: Clinical Trial PI. Liu:Pairwise Plants: Consultancy, Patents &amp; Royalties; Editas Medicine: Consultancy, Patents &amp; Royalties; Beam Therapeutics: Consultancy, Patents &amp; Royalties; Prime Medicine: Consultancy, Patents &amp; Royalties. Weiss:Beam Therapeuticcs: Consultancy, Current equity holder in private company; Esperion Therapeutics: Consultancy, Current equity holder in private company; Novartis: Consultancy, Current equity holder in private company; Cellarity Inc.: Consultancy, Current equity holder in private company; Rubius Inc.: Consultancy, Current equity holder in private company.

Base Editing Eliminates the Sickle Cell Mutation and Pathology in Hematopoietic Stem Cells Derived Erythroid Cells

Sickle cell disease (SCD) is a chronic, life-altering multisystem disorder that affects millions of individuals worldwide. Strategies for genetic therapy of autologous SCD hematopoietic stem cells (HSCs) include lentiviral vector (LV) delivery of an anti-sickling β-like globin gene and genome editing, either to revert the SCD mutation by homology-directed repair (HDR) or to induce the expression of fetal hemoglobin (HbF, α2γ2) in red blood cell (RBC) by non-homologous end-joining repair (NHEJ). Base editing is a newer technology that offers the potential for facile generation of more precise genetic alterations with improved safety features. Adenosine base editors (ABEs) convert A-T base pairs to G-C pairs at loci targeted by a single guide (sg) RNA and Cas9 nickase. In contrast to LV vectors and standard Cas9 genome editing, ABEs function through mechanisms that are independent of double-stranded DNA breaks (DSBs), which can cause large deletions, structural DNA rearrangements and TP53-mediated DNA damage responses leading to cell death or malignant transformation. Base editors cannot generate the T-to-A transversion required to revert the mutant SCD codon (Val, GTG) to wild-type (Glu, GAG). However, ABEs can convert the Val codon to Ala (GCG) to generate the naturally occurring, non-sickling variant hemoglobin "Makassar" (HbG). Hemoglobin Makassar heterozygotes and 1 reported homozygote exhibit normal RBC indices, indicating that the variant is benign. We used protein directed evolution to generate a new ABE (ABE8e-NRCH) that accesses a nearby CACC PAM to convert HbS alleles to HbG efficiently in heterologous cells. To demonstrate therapeutic proof of concept, we electroporated ABE8e-NRCH mRNA and targeting sgRNA or ABE8e-NRCH/sgRNA ribonucleoprotein (RNP) complex into 3 different SCD donor CD34+ hematopoietic stem and progenitor cells (HSPCs). After 48 hours, conversion of the HbS allele to HbG was 58±5% with ABE8e-NRCH mRNA/sgRNA and 34±5% with RNP (n=3). On target editing was maximal at 144 hours: 80±2% with ABE8e-NRCH mRNA/sgRNA and 44±7% with ABE8e-NRCH protein (n=3). The indel rate resulting from inadvertent DSBs was &amp;lt;1%, and missense bystander mutations were not detected. In vitro differentiation of base-edited CD34+ cells generated late stage erythroid cells with 76% HbG/14% HbS protein after editing with ABE8e-NRCH mRNA/sgRNA, and 52% HbG/37% HbS after editing with RNP. The sickling rate of in vitro-generated reticulocytes exposed to hypoxia (2% O2) was 52% in controls derived from unedited CD34+ cells, 16% after editing with ABE8e-NRCH mRNA/sgRNA and 27% after editing with RNP. To assess base editing in repopulating HSCs, we transplanted ABE8e-NRCH-edited SCD CD34+ cells into NBSGW mice. The editing frequency before transplantation was ~64% for ABE8e-NRCH mRNA/sgRNA and 40% for RNP at 48 hours after electroporation. Mice were euthanized after 16 weeks and base editing was analyzed in human donor-derived cells. Overall editing rates were preserved in repopulating cells: 68%±3% for ABE8e-NRCH mRNA/sgRNA and 36%±3% for RNP (n=4). The editing frequencies were similar in donor cell-derived myeloid, erythroid, and B cell-lineages, indicating that base editing did not alter hematopoietic development. Bone marrow erythroblasts derived from base-edited and control CD34+ HSPCs exhibited similar maturation profiles and enucleation. Erythroblasts generated in vivo from HSPCs from SCD patient exhibited potentially therapeutic levels of HbG protein: 58±3% with ABE8e-NRCH mRNA/sgRNA and 27%±3% with RNP (n=4). Adenine base editor conversion of the HbS allele to the Makassar variant in autologous HSCs represents a new therapeutic approach for SCD. Disclosures Yen: Beam Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Henderson:Trilink Biotech: Current Employment. Mccaffrey:Trilink Biotech: Current Employment. Liu:Pairwise Plants: Consultancy, Patents &amp; Royalties; Prime Medicine: Consultancy, Patents &amp; Royalties; Editas Medicine: Consultancy, Patents &amp; Royalties; Beam Therapeutics: Consultancy, Patents &amp; Royalties. Weiss:Rubius Inc.: Consultancy, Current equity holder in private company; Cellarity Inc.: Consultancy, Current equity holder in private company; Novartis: Consultancy, Current equity holder in private company; Esperion Therapeutics: Consultancy, Current equity holder in private company; Beam Therapeuticcs: Consultancy, Current equity holder in private company.

Control of Fetal Hemoglobin Levels By NFI Transcription Factors

Sickle cell disease and some forms of b-thalassemia are disorders, which can be improved by therapies that elevate HbF, the fetal form of hemoglobin. It is currently thought that in adult type erythroid cells, silencing of the fetal type b-globin-like genes HBG1 and HBG2 is accomplished predominantly by two major transcription factors: BCL11A and ZBTB7A. However, it is unknown whether there are additional transcription factors that contribute to the repression of the HBG1/2 genes. Here, using a DNA-binding domain focused CRISPR-Cas9 screening approach, we identified NFIA as a novel regulator of HbF expression. NFIA belongs to the NFI transcription factor family, which is composed of NFIA, NFIB, NFIC and NFIX. NFIA and NFIX are the predominantly expressed forms in human erythroid cells, and their expression is higher in adult erythroblast cells when compared to fetal erythroid cells. CRISPR-Cas9 mediated disruption of NFIA in an immortalized human umbilical cord blood-derived erythroid progenitor cell line 2 (HUDEP2) or CD34+ hematopoietic stem and progenitor cell-derived primary erythroblast cells led to a modest reactivation of HBG1/2 mRNA expression, whereas disruption of NFIX had little effect. However, combined NFIA and NFIX disruption produced a substantial increase in HBG1/2 expression, suggesting that these factors function in a partially redundant manner. ChIP-seq and RNA-seq studies showed that NFIA and NFIX have comparable chromatin binding and activity profiles in human erythroid cells. ChIP-seq failed to detect NFI protein occupancy at or near the HBG1/2 genes. However, given the known difficulty in detecting repressor molecules by ChIP at the silent HBG1/2 genes [Martyn et al., Nature Genetics 2018; Liu et al., Cell 2018], we tested a direct involvement of NFI proteins by disrupting putative NFI binding sites near the HBG1/2 genes. Perturbation of one such NFI motif residing upstream of the transcription start site in the HBG1/2 promoters both in HUDEP2 and primary human erythroid cells markedly increased HBG1/2 mRNA levels, comparable to those achieved by combined disruption of NFIA and NFIX. Mutation of another putative NFI motif within intron1 in the HBG1/2 gene also significantly raised HbF levels. While these results implicate NFI proteins in the direct silencing of the HBG1/2 genes, the identity of the bound factors at the NFI motifs remains to be established. Studies are currently ongoing that use alternative approaches such as Cleavage Under Target &amp; Release Using Nuclease (CUT &amp; RUN) to map the chromatin occupancy of NFI factors at the HBG1/2 genomic region. We will also discuss results from ongoing studies of NFI factors in NBSGW mice models. In sum, we uncovered NFI transcription factors as a novel HbF regulator suggesting that the silencing of HbF involves a transcription factor network that is more complex than previously appreciated. Disclosures Weiss: Rubius Inc.: Consultancy, Current equity holder in private company; Cellarity Inc.: Consultancy, Current equity holder in private company; Novartis: Consultancy, Current equity holder in private company; Esperion Therapeutics: Consultancy, Current equity holder in private company; Beam Therapeuticcs: Consultancy, Current equity holder in private company. Blobel:Pfizer: Research Funding; Fulcrum Therapeutics: Consultancy.

Correction of the Energetic Defects in Pyruvate Kinase Deficiency through Genome Editing in Hematopoietic Stem and Progenitor Cells

Pyruvate kinase deficiency (PKD) is an autosomal recessive disorder caused by mutations in the PKLR gene that lead to a reduction of the erythroid pyruvate kinase (RPK) protein activity, which causes an energetic imbalance in PKD erythroid cells. This disease is associated with reticulocytosis, splenomegaly and iron overload, and may be life-threatening in severely affected patients. In selected cases, allogeneic Hematopoietic Stem Cell Transplantation has been shown to correct the disorder. Therefore, autologous HSCT of genetically corrected cells will offer a durable and curative therapeutic option. In fact, a global Phase I clinical trial (clinicaltrials.gov #NCT04105166) is underway to evaluate the feasibility and safety of lentiviral mediated gene therapy with severe PKD. Looking for a guided integration of the exogenous therapeutic DNA sequences, herein we conducted a gene editing approach to correct PKD in human Hematopoietic Stem Cells (HSCs). We developed a knock-in approach to insert either a TurboGFP expression cassette or a promotor-less therapeutic codon optimized RPK cDNA (coRPK) at the genomic starting site of the PKLR gene, a gene editing strategy that will correct most of the mutations present in PKD patients. This gene editing approach combines RNP nucleofection and adeno-associated viral vector (AAV6) mediated delivery of homologous donors. In order to assess the safety of the proposed gene editing approach, we performed GUIDE-Seq and rhAmpSeqTM analyses of different single guide RNAs targeting the PKLR starting site. We found PKLR sgRNAs that showed a high targeting efficiency, up to 40% targeted hematopoietic progenitors (in vitro semisolid colony forming units) and a safety profile, with no off-targets detected above threshold values (0.1%) and in the absence of cellular toxicity, when applied to healthy cord blood CD34+ (CB-CD34+) cells. These gene-edited CB-CD34+ cells engrafted efficiently in both primary and secondary immunodeficient NSG recipient mice, demonstrating the gene editing in long-term hematopoietic repopulating HSCs. Furthermore, we evaluated the therapeutic potential of this gene editing strategy to restore the energetic imbalance in erythroid cells derived from PKD patients. CD34+ cells from 4 different PKD patients, were purified, nucleofected with PKLR sgRNA and transduced with AAV-coRPK donor. In vitro differentiated erythroid cells derived from edited PKD CD34+ expressed coRPK mRNA and restored their energetic defect up to normal values obtained in in vitro differentiated erythroid cells from healthy donor cells. Moreover, gene edited mobilized Peripheral Blood CD34+ (mPB-CD34+) cells from a PKD patient engrafted efficiently in immunodeficient NBSGW mice, having human cells that showed the specific integration of coRPK donor 2 months post-transplant. Overall, these results demonstrate the feasibility and safety of PKLR gene editing in human HSPCs and, therefore, its potential clinical application for the treatment of PKD patients. Disclosures Dever: Integral Medicines: Current Employment. Turk:Integrated DNA Technologies, Inc. (IDT): Current Employment, Current equity holder in publicly-traded company. Bianchi:Agios Pharmaceuticals: Other: Scientific Advisor. Behlke:Integrated DNA Technologies, Inc. (IDT): Current Employment, Current equity holder in publicly-traded company. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from this company., Patents &amp; Royalties, Research Funding. Segovia:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from the Company., Patents &amp; Royalties, Research Funding.

Robust Pre-Clinical Results and Large-Scale Manufacturing Process for Edit-301: An Autologous Cell Therapy for the Potential Treatment of SCD

Sickle cell disease (SCD) is an inherited blood disorder affecting approximately 100,000 individuals in the United States. As fetal hemoglobin (HbF) has been shown to be protective against clinical manifestation of SCD, we are developing EDIT-301, an autologous cell therapy comprising CD34+ cells genetically modified using a Cas12a ribonucleoprotein (RNP) to promote HbF expression to treat SCD. Fetal hemoglobin induction for EDIT-301 is achieved by disrupting the HBG1 and HBG2 promoter distal CCAAT-box region where naturally occurring mutations are found to be associated with elevated HbF expression. Cas12a was selected over Cas9 due to the more productive and sustainable (NHEJ derived) indel profile, as well as high specificity. Using Cas12a RNP, on-target editing of ~90% was achieved in mobilized peripheral blood CD34+ cells (mPB-CD34+ cells) from both healthy and SCD donors at research scale with no detectable off targets. Editing of CD34+ cells led to an average of 43% and 54% of HbF expression in the erythroid progeny of normal donor and SCD donor cells respectively in a pancellular fashion (~93% population). The robust HbF induction in SCD red blood cells (RBCs) resulted in significant phenotypic and functional improvement including reduced sickling and increased deformability under hypoxia ex vivo. Using a microfluidic assay that replicated blood flow in microvasculature under varying oxygen conditions, SCD RBCs derived from RNP electroporated CD34+ cells showed improved rheological behavior. The rheology improvement under hypoxia was strongly correlated with the increased levels of HbF in each sample. Infusion of the modified CD34+ cells from normal donors into NBSGW mice resulted in long-term multi lineage and polyclonal reconstitution. Editing levels at 16 weeks post infusion were &amp;gt; 90% in all human lineages tested, demonstrating the efficient editing of SCID-repopulating hematopoietic stem cells (HSCs). Consistent with the high editing levels, human erythroid cells from the bone marrow of mice that received Cas12a-RNP treated cells demonstrated pancellular (~90% F+ RBCs) HbF expression averaging 40-50% of total hemoglobin compared to ~5% HbF observed in the control group. We have developed a consistent large-scale process using functionally closed, semi-automated systems suitable for use in clinical manufacturing. We have shown robust editing of normal donor CD34+ cells and SCID-repopulating HSCs with the clinical scale process. Editing levels of &amp;gt;90% were detected after long term engraftment in mice. In summary, we have demonstrated successful on-target editing of mPB CD34+ cells derived from both normal and SCD donors using a Cas12a RNP, which coincided with robust HbF induction and a phenotypic reduction of sickling in the SCD erythroid progeny, as well as improved rheological behavior. Editing of the HBG1 and HBG2 promoters using this RNP was highly specific with no measurable off-target. In vivo, cells from normal donors readily engrafted and reconstituted all blood cell lineages at levels comparable to unedited cells. Finally, a robust large-scale manufacturing process has been developed to supply material for the clinical setting. Based on these results, we are completing the activities required to assess EDIT-301 in the clinic as treatment for SCD. Disclosures De Dreuzy: Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Heath:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Sousa:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Janoudi:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. An:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Albright:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Teixeira:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Monesmith:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Zhang:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company. Chang:Editas Medicine Inc.: Current Employment, Current equity holder in publicly-traded company.

Modelling Congenital Dyserythropoietic Anemia Type II through Gene Editing in Hematopoietic Stem and Progenitor Cells

Congenital dyserythropoietic anemias (CDAs) are a group of inherited anemias that affect the development of the erythoid lineage. They are characterized by ineffective erythropoiesis with distinct morphologic abnormalities of erythroblasts, a degree of hemolysis, and secondary hemochromatosis. Patients usually present with congenital anemia, jaundice, splenomegaly, and an absolute reticulocyte count inadequate for the degree of anemia. CDA type II (CDAII) is the most frequent type. CDAII patients show anemia of variable degrees, and 20% are transfusion dependent. The most specific finding of CDAII marrow is the presence of more than 10% mature binucleated erythroblasts with two nuclei at the same erythroid maturation stage. Treatment of CDAII patients may involve blood transfusions, iron chelation therapy and splenectomy. The only described definitive therapy is allogeneic bone marrow transplantation, which implies additional side effects for these patients. Therefore, new therapeutic approaches are needed. CDA II is caused by mutations in the SEC23B gene. SEC23B is part of coat protein complex II (COPII). COPII is involved in protein processing and Golgi-reticulum trafficking. However, how mutations in SEC23B cause CDA II is not known yet. Therefore, studying the role of SEC23B in the erythropoiesis will help to elucidate the underlying mechanism of CDA II and to develop new therapeutic approaches for the disease. We have developed a CDA II model in human cells through the introduction of genomic mutations in the gene using the CRISPR/Cas9 gene editing system. Different single guides RNAs (sgRNA) targeting the start of the coding sequence of human SEC23B gene were designed and tested in human erythroleukemia K562 cell line and in healthy human cord blood hematopoietic stem and progenitors (hCB-CD34+). The gene editing outcome at SEC23B gene was assessed at: i) genomic level through Sanger sequencing, Inference of CRISPR Edits (ICE) and Next-Generation Sequencing (NGS). ii) Protein level through Western-blot analysis. iii) Functional level through morphological analysis and erythroid differentiation either in vitro or in vivo in human hematopoietic chimeras in NOD.Cg-KitW-41JTyr+PrkdcscidIl2rgtm1Wjl/ThomJ (NBSGW) mice. K562 cells were nucleofected with three different sgRNAs, as ribonucleoprotein (RNP), independently or in combination. Afterwards, seventy five K562 clones were established from the cells nucleofected with the most efficient sgRNA or with the combination of the three sgRNAs. Forty per cent of them showed a high efficiency of knock-out (higher than 50% of alleles). Eight SEC23BKO clones were selected for further analysis. All of those eight clones showed a reduction in SEC23B protein and six of them had a lower proliferation than control cells and morphological abnormalities, such as presence of bi/multinucleated cells. Moreover, when CB-CD34+ cells were nucleofected with the most efficient sgRNA or with the combination of the three sgRNAs, up to 80% of knock-out efficiency and close to 90% reduction of SEC23B protein were obtained. Interestingly, when those gene edited hematopoietic progenitors were differentiated in vitro to erythroid cells, their terminal differentiation was hampered, with a reduce percentage of enucleated cells and the presence of high number of bi/multinucleated cells. Similarly, the in vivo erythroid differentiation of these gene edited progenitors two months after HSPC transplant into NBSGW mice showed again an impairment of terminal erythroid differentiation with an increment in the percentage of erythroid bi/multinucleated cells without altering other human hematopoietic lineages. In summary, CRISPR/Cas9 system has been used to model CDA II in a human cell line and in human hematopoietic progenitors through the knock-out of SEC23B gene. Our system reproduced the most relevant feature characteristic of CDA II pathology. This gene editing based CDA II model will allow the study of how mutations in SEC23B cause CDA II and the development of new therapeutic strategies to cure this disease. Disclosures Tornador: Bloodgenetics: Current Employment. Sanchez:Bloodgenetics: Current Employment. Segovia:Rocket Pharmaceuticals, Inc.: Consultancy, Current equity holder in publicly-traded company, Other: Consultant for Rocket Pharmaceuticals, Inc. and has licensed medicinal products and receives research funding and equity from the Company., Patents &amp; Royalties, Research Funding.

REPOPULATION OF FUNCTIONAL MULTILINEAGE HUMAN HAEMATOPOIETIC CELLS IN NON-IRRADIATED NBSGW MICE

Introduction: Humanised mouse models provide a platform to investigate human immune biology in vivo, reducing the need for large animal studies. Importantly they can overcome biological differences between humans and other species, reducing the uncertainty in progressing to clinical studies. In transplantation research, humanised immune system (HIS) models have provided pre-clinical evidence for human-specific therapies such as biologics and regulatory T cell therapy, which are now in clinical trials. Most HIS models lack multilineage leukocyte development, limiting accurate representation of the full alloresponse in transplantation. To address this, we assessed immune reconstitution and human skin transplant rejection in NOD,B6.SCID Il2rγ−/− KitW41/W41 (NBSGW) mice, which are capable of long-term multilineage haemopoiesis without irradiation due to the presence of an inactivating mutation in the stem cell growth factor receptor. Materials and Methods: Human cord blood CD133+ haematopoietic stem cells (HSCs) were injected intravenously into non-irradiated NBSGW mice (n=20). Engraftment in blood and lymphoid organs was assessed over 20 weeks. In a separate group, HSC-engrafted mice (HIS-NBSGW) underwent allogeneic human skin transplantation 10-12 weeks post-injection (Figure 1).At the point of skin transplant rejection (or after 100 days) blood, spleen, bone marrow (BM), thymus and skin were analysed by multiparameter flow cytometry and NanoString gene expression analysis. Results and Discussion: High levels of human CD45+ cell chimerism developed reliably in the blood, BM and spleen after HSC transplantation in a dose-dependent manner (Figure 2).Multilineage engraftment was achieved, consisting of human myeloid cells, B and T lymphocytes at multiple recognised stages of development. Expression of Human Leukocyte Antigen (HLA) Class I and II molecules was identified on CD3- thymocytes. Antigen-presenting cells (APCs), B cells and T cells were found to be functional in in vitro assays. In mice with multilineage human leukocyte reconstitution, allogeneic human skin grafts rejected. Rejection was associated with RNA markers of upregulated immune function and elevated production of IgM, IgG and Th1, Th2 and Th17-related cytokines (Figure 3).Conclusion: The HIS-NBSGW model provides a technically straightforward research system utilising adult mice, which does not require irradiation, co-transplantation of human haematopoietic stromal tissues, or provision of human cytokines. By producing functional multilineage immune cells, it is a useful method for assessing HSC function in vivo and a framework for assessing human transplant responses encompassing the breadth of innate, cellular and humoural immunity. Clarendon Fund, University of Oxford. MRC Discovery Award, Ref: MC_PC_15069.

3117 – GPRASP2 REGULATES CXCR4 STABILITY AND SIGNAL TRANSDUCTION IN HUMAN HEMATOPOIETIC CELLS WITH IMPLICATIONS FOR HSPC TRANSPLANTATION

Transplantation of hematopoietic stem and progenitor cells (HSPCs) is a life-saving therapy that is limited by donor availability and low cell numbers in sources such as cord blood. Advances in our understanding of the biochemical pathways that determine HSPC engraftment will allow for the optimization of transplantation strategies. We previously found that knockdown of GPRASP (G-protein Coupled Receptor Associated Sorting Protein) protein family members increases CXCR4 abundance and transplantation efficiency in murine HSPCs. Here, we demonstrate that GPRASP2 facilitates the acute agonist-induced degradation of CXCR4 in human hematopoietic cells and limits signaling downstream of CXCR4. Initial data also indicate that loss of GPRASP2 may increase the transplantation efficiency of human CD34+ cells. An antibody protection assay performed in HL60 cells demonstrated that knockdown of GPRASP2 significantly increases stability of CXCR4 following SDF-1 treatment. Phosphoflow experiments were performed to quantify the impact of GPRASP2 on signaling pathways downstream of CXCR4 including AKT, ERK1/2, p38, and JAK/STAT. SDF-1 treatment primarily resulted in phosphorylation of ERK1/2 and this was significantly increased by GPRASP2 knockdown. To test the role of GPRASP2 in human HSPC transplantation, CD34+ cells were edited by CRISPR/Cas9 with sgRNAs targeting GPRASP2 or the AAVS1 safe harbor site and then xenotransplanted into NBSGW mice. Initial results indicate that GPRASP2 KO results in higher human chimerism than is seen for AAVS1-targeted cells. In addition to assessing chimerism between transplants, changes in INDEL frequency within each transplant will be tracked for 16 weeks to determine the fitness of GPRASP2 knockout cells as compared to non-edited cells. This work reveals novel aspects of CXCR4 regulation by GPRASP2 and illuminates a potential point of intervention for the optimization of HSPC transplantation.

Early T Cell Activation Metrics Predict Graft-versus-Host Disease in a Humanized Mouse Model of Hematopoietic Stem Cell Transplantation.

Acute graft-versus-host disease (GVHD) is a frequent complication of hematopoietic transplantation, yet patient risk stratification remains difficult, and prognostic biomarkers to guide early clinical interventions are lacking. We developed an approach to evaluate the potential of human T cells from hematopoietic grafts to produce GVHD. Nonconditioned NBSGW mice transplanted with titrated doses of human bone marrow developed GVHD that was characterized by widespread lymphocyte infiltration and organ pathology. Interestingly, GVHD was not an inevitable outcome in our system and was influenced by transplant dose, inflammatory status of the host, and type of graft. Mice that went on to develop GVHD showed signs of rapid proliferation in the human T cell population during the first 1-3 wk posttransplant and had elevated human IFN-γ in plasma that correlated negatively with the expansion of the human hematopoietic compartment. Furthermore, these early T cell activation metrics were predictive of GVHD onset 3-6 wk before phenotypic pathology. These results reveal an early window of susceptibility for pathological T cell activation following hematopoietic transplantation that is not simply determined by transient inflammation resulting from conditioning-associated damage and show that T cell parameters during this window can serve as prognostic biomarkers for risk of later GVHD development.